Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • A61K38/1719—Muscle proteins, e.g. myosin or actin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/56—Porous materials, e.g. foams or sponges
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/58—Materials at least partially resorbable by the body
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/60—Materials for use in artificial skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

• the invention is directed to methods and compositions useful for wound healing and tissue regeneration.
• Wound repair involves complex biological processes, and managing wounds with a large surface area is a great challenge (Singer, A. J. & Clark, R. A. Cutaneous wound healing. The New England journal of medicine 341, 738-746 (1999), Passier, R., van Laake, L. W. & Mummery, C. L. Stem-cell-based therapy and lessons from the heart. Nature 453, 322-329 (2008)).
• Over 100 million patients in the industrialized world suffer from wounds every year (Takeo, M., Lee, W. & Ito, M. Wound healing and skin regeneration. Cold Spring Harbor perspectives in medicine 5, a023267 (2015)).
• materials/agents should promote reepithelialization and wound healing without inducing neoplasm development, minimize pain, decrease the risk of infection, and reduce cosmetic deformity; the selection of such materials is a major component of modern wound management and regenerative medicine (Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature biotechnology 23, 47-55 (2005)).
• the native ECM possesses biophysical properties of surface topology, bulk stiffness, elasticity, shear force, and pore size that are important for cue-guided cell migration and stem cell differentiation.
• the ECM also anchors diverse soluble growth factors, signal receptors and adhesion molecules, which influence cell fates.
• Reconstitued ECM and ECM- derived materials may lack the native topology information, soluble growth factors and anchored factor concentrations.
• large-area (ft 2 -scale) native biomaterials containing the complete and native ECM biophysical, biochemical, and biomechanical properties that make them ideal materials for use in wound repair are currently not available (Chien, K. R. Regenerative medicine and human models of human disease. Nature 453, 302-305 (2008)).
• Wound infection continues to be a challenging problem and represents a considerable healthcare burden.
• the physical barrier at wounds to prevent micro-organism contamination and colonization is essential for wound infection prevention.
• Wounds (acute or chronic) usually contain micro-organisms, including bacteria, fungus and virus (Sood, A., Granick, M. S., and Tomaselli, N. L. (2014) Wound Dressings and Comparative Effectiveness Data. Advances in wound care 3, 511-529, Lall, R. R., Wong, A. P., Lall, R. R., Lawton, C. D., Smith, Z. A., and Dahdaleh, N. S. (2014) Evidence-based management of deep wound infection after spinal instrumentation.
• Bacteria may result in spreading infection in nearby tissues or systemic infection which presents systemic illness.
• Acute wounds include surgical and traumatic wounds, and burns ( Palmieri, T. L., Przkora, R., Meyer, W. J., 3rd, and Carrougher, G. J. (2014) Measuring burn injury outcomes.
• Chronic wounds include diabetic foot ulcers, venous leg ulcers, arterial leg/foot ulcers and pressure ulcers (Moran, M. E. (2014) Scleroderma and evidence based non-pharmaceutical treatment modalities for digital ulcers: a systematic review. Journal of wound care 23, 510-516, Baltzis, D., Eleftheriadou, I., and Veves, A.
• Native cell cytoplasm membrane is a bi-lipid membrane harboring thousands of membrane proteins modified by phosphor, sugar chains, and so on.
• Cell cytoplasm membrane can effectively form physical barriers for micro-organism infection (Hahler, B. (2006) Surgical wound dehiscence. Medsurg nursing : official journal of the Academy of Medical-Surgical Nurses 15, 296-300; quiz 301).
• the area of cytoplasm membrane of a single cell is too small ( ⁇ 2 level) to be applied for clinical usage.
• the fragility and tiny thickness (5 ⁇ 10nm) of cell membrane make it difficult to collect cytoplasm membranes of multiple cells and reorganize them into a useful membrane for wound care. There remains a need for large-area membranes useful for wound repair and effective prevention and management of wound infection.
• the present disclosure addresses this need and is based on the discovery that native primary human epithelial cells grown in matrix support produce large-area microfilament network.
• the microfilament network functions as physical barrier for prevention and management of wound infection, including chronic and acute wounds, burn care, acute and surgical wound care.
• normal primary human epithelial cells cultured on cell matrix generate a large-area of microfilament network.
• the microfilament network is processed to remove the cells or nuclei.
• the microfilament network can be applied to wounds and acts as physical barrier to prevent micro-organism induced wound infection.
• the engineered large area microfilament network-matrix complex layer can effectively prevent infection by microorganisms, including bacteria, fungi and viruses.
• Microfilaments the main cytoskeletal polymers in eukaryotic cells, are polymerized by actin subunits and actin-binding proteins. Microfilaments are essential for cell division and cytokinesis, cell shape maintenance, vesicle transportation, signal transduction and cell motility. Most animal cells maintain a micrometer ⁇ m)-scale cell size, which restricts the area of the cytoskeletal microfilament networks to the same scale. According to certain aspects, human epithelial cell masses cultured in vitro on matrigel generate super large extracellular microfilament networks (EMNs). Such EMNs facilitate cell migration. According to one aspect, these EMNs can grow to square foot (ft 2 ) size.
• EFNs extracellular microfilament networks
• these microfilament networks have utility in a general wound healing therapy.
• the EMNs generated by human epithelial cell masses contain extensive membrane- enclosed microfilaments.
• the microfilament network is treated to remove cell masses to produce artificial, cell-less, and ft 2 -scale, ultra-large multi-layered lattices (UMLs).
• UMLs can be used to facilitate the reepithelialzation.
• these UMLs have utility for healing of second degree thermal burn wounds in mice.
• the EMNs produced by human epithelial cells promote/facilitate cell migration.
• the UMLs obtained after cell removal can be used to facilitate wound healing and tissue regeneration.
• embodiments of the present disclosure are directed to networks including cell-derived microfilaments interconnected in a continuous lattice or mesh structure between a plurality of microfilament source regions.
• embodiments of the present disclosure are directed to methods of producing a network of microfilaments including the steps of: culturing cells in a matrix support and cell culture medium wherein the cells proliferate and form aggregated cell masses, and wherein the cell masses produce microfilaments external to and surrounding the cell masses, and wherein the extracellular microfilaments connect and form a continuous extracellular microfilament network.
• the network of microfilaments is treated to remove nuclei of the cells and/or the cells from the network.
• the cell masses form on top of the matrix support.
• the microfilaments of the network are extracellular microfilaments. In another embodiment, the microfilaments are membrane-enclosed. In one embodiment, the microfilaments include actin. In another embodiment, the actin includes ⁇ -actin. In one embodiment, the microfilaments are about 1-1000 ⁇ in length. In another embodiment, the microfilaments are branched. In yet another embodiment, the microfilaments have about 2-10 branches. In one embodiment, multiple microfilaments align together and form bundles of diverse architectural structures. In another embodiment, the microfilament source regions form connection nodes for the continuous lattice or mesh structure. In one embodiment, the network further includes adhesive materials.
• the adhesive materials associate with the microfilaments and enlarge the diameter of the microfilaments.
• the network has an area in the range of about 1 ⁇ 2 to about 500 cm 2 and a thickness in the range of about 1 nm to about 0.5 cm.
• the network is single or multiple layered.
• the surface area of the microfilaments is greater than an equivalent unit of an intra-cellular cytoskeletal microfilament network surface area.
• the network is porous.
• the pore size ranges from about 0.1 - 5 ⁇ in diameter.
• the network further includes bioactive and/or bioinactive agents.
• the bioactive agents are therapeutic drugs.
• the microfilament source regions include cells.
• the network is present on a matrix support.
• the matrix support is biodegradable.
• the network is present on a Matrigel matrix support.
• the network lacks nuclei from cells.
• the network lacks cells.
• the microfilament source regions include eukaryotic cells with or without genetic modification.
• the eukaryotic cells are mammalian cells.
• the mammalians cells are human cells.
• the human cells are human mammary epithelial cells.
• the microfilaments are embedded within the top surface of the matrix support.
• the matrix is a Matrigel.
• the matrix support inhibits cell attachment and migration.
• the present disclosure provides a method for treating a medical condition via applying the microfilament network of to an area in need of treatment.
• the microfilament network is applied with the matrix.
• the microfilament network is applied without the matrix.
• the medical condition is a wound or an injured tissue.
• the microfilament network facilitates healing and/or prevents infection of the wound or the injured tissue.
• the medical condition is a burn.
• the method includes enhancing or promoting re-epithelialization of damaged skin.
• the method further includes administering therapeutic drugs prior to, concurrent with or after applying the microfilament network to the area in need of treatment.
• the present disclosure contemplates a method of producing a continuous network of cell-derived microfilaments in vitro including: culturing a plurality of cell clusters on a surface of a matrix substrate under conditions such that the cell clusters produce microfilaments external to and surrounding the cell clusters, and where the extracellular microfilaments connect and form a continuous extracellular network of microfilaments between the cell clusters on the surface of the matrix substrate.
• the cell clusters are spaced apart by an average distance of between about 1 microns and about 1000 microns.
• the microfilaments connecting the cell clusters have an average length of between about 1 microns and about 1000 microns.
• the continuous extracellular microfilament network is a multilayered lattice. In another embodiment, the microfilaments are branched. In one embodiment, the continuous extracellular microfilament network is a multilayered lattice including long microfilaments having a length of between about 10 microns and about 1000 microns and short microfilaments having a length of between about 1 microns and about 10 microns. In another embodiment, the continuous extracellular network of microfilaments has a surface area of between about 0.01 cm 2 and 500 cm 2 . In one embodiment, the continuous extracellular microfilament network is a mesh. In another embodiment, the continuous extracellular microfilament network is porous.
• cells are implanted on the surface of the matrix substrate and where the cells migrate and aggregate into preclusters and proliferate to form the cell clusters.
• the method further includes removing cell nuclei or the cell clusters from the continuous extracellular microfilament network.
• the matrix substrate inhibits attachment and/or migration of the cells.
• the method further includes separating the matrix substrate from the continuous extracellular microfilament network.
• a method for facilitating wound repair and/or tissue regeneration comprising applying the microfilament network to a site in need thereof.
• the microfilament network is applied with or without the matrix.
• the microfilament network forms square foot (ft 2 )-scale ultra-large microfilament lattices (UMLs).
• UMLs construct an environment for cell migration.
• the microfilament networks are multilayered and three dimensional (3D).
• the method includes removing cell masses and producing acellular UMLs (AUMLs).
• the AUMLs facilitate wound repair.
• the method includes applying the AUMLs to a wounded site.
• applying the AUMLs allows keratinocytes to engender large tunnels in the reepithelialized epidermis.
• the large tunnels provide pathways for cells and nutrients to the site of wound repair.
• the microfilament network prevents infection of the wounded site.
• method includes enhancing or promoting re-epithelialization of wounded skin.
• the wound is second degree thermal burn wounds.
• method further includes administering therapeutic drugs prior to, concurrent with or after applying the microfilament network to the area in need of treatment.
• FIGS. 1A-F show images of human mammary epithelial cells (HMECs) forming masses on the Matrigel matrix.
• FIG. 1 A depicts that the individual human mammary epithelial cells (HMECs) in the 2D environment present as irregular shapes shapes (left) while consistently display a spherical morphology in the 3D (three-dimensional) Matrigel culture on the (right).
• HMECs were implanted on the thick Matrigel matrix (20 ⁇ 30 ⁇ in depth) at a low cell density (lxlO 3 cells per well in 6-well-plates). The inverted phase contrast images were taken at 5 hours (h) after cell implantation.
• FIG. 1 A depicts that the individual human mammary epithelial cells (HMECs) in the 2D environment present as irregular shapes shapes (left) while consistently display a spherical morphology in the 3D (three-dimensional) Matrigel culture on the (right).
• HMECs were implanted on the thick Matrigel matrix
• IB is a representative toluidine blue image showing a cross section of a spherical HMEC with a minimal surface area exposed to the Matrigel matrix (MM).
• FIG. 1C is a representative toluidine blue image of a cross section of a cell group showing that two spherical stacked HMECs (white asterisks) have no surface contact with the Matrigel matrix (MM).
• FIG. ID is a representative toluidine blue image of a cross section of a mounded cell mass showing that multiple stacked HMECs (white asterisks) are not in contact with the Matrigel matrix (MM).
• FIG. IE is a representative inverted phase contrast image showing that the HMECs form many cell masses after cell migration, aggregation, proliferation and stacking.
• FIGS. 2A-B show fluorescent images that human cell masses generate superlarge and continuous extracellular microfilament networks.
• FIG. 2A shows fluorescence microscopy examination of the composition and architecture of the extracellular microfilament networks. Plasmids encoding enhanced green fluorescence protein (EGFP) tagged-plasma membrane Ca 2+ -ATPase2 (EGFP-PMCA2) were transiently transfected into primary normal human mammary epithelial cells (HMECs). HMECs with EGFP-PMCA2 overexpression were transplanted onto the Matrigel matrix surface and cultivated for 64 hours (h). HMECs migrated, aggregated, proliferated, and formed cell masses (CMs) on the Matrigel layers.
• CMs formed cell masses
• DAPI (4',6-diamidino-2-phenylindole) staining image shows that there are no cell nuclear materials between and around the cell masses.
• Middle panel the cell masses generate a large quantity of membrane -enclosed, long, branched extracellular microfibers (ECMFs, white arrows) exterior to the cell masses.
• the nested microfibers form large networks surrounding the cell masses.
• the long extracellular microfibers (bold white arrows) connect the two cell masses (CMl and CM2).
• CMl and CM2 right panel: the merged image shows that the nested extracellular microfiber networks are located exterior to the cell masses that generate them.
• FIG. 2B shows fluorescent images of actin-composed microfilaments.
• CM-PMCA2 actin- based and membrane-enclosed extracellular microfilaments
• ECMFs actin- based and membrane-enclosed extracellular microfilaments
• the extracellular microfilaments form networks and bundles (yellow arrows).
• An extracellular microfilament connection node EMCN, purple arrows
• an extracellular microfilament assembled network space EMNS, white asterisks
• microfilament adhesive materials AMs
• Distribution of the lengths of a total of 385 randomly chosen HMEC extracellular microfilaments approximately 51% are in the range of 1-5 ⁇ , 22.6% in the range of 5-10 ⁇ , 8.9% in the range of 10-20 ⁇ , 5.2% in the range of 20-40 ⁇ , 2% in the range of 40-100 ⁇ , 1% in the range of 100-300 ⁇ , and 2% reach up to 1000 ⁇ .
• FIGS. 4A-E show fluorescent images and bar graphs of development of the extracellular microfilament networks. Representative fluorescence images of the HMEC extracellular microfilament network development along time. HMECs with EGFP-PMCA2 overexpression were transplanted onto the Matrigel matrix surface and cultivated for a period of time.
• FIGS. 5A-C show images of the architectural structures of extracellular microfilament networks.
• FIG. 5A shows a merged fluorescence image that extracellular microfilaments (ECMF, red arrows) connect via extracellular microfilament connection nodes (EMCN, purple arrows) with regularly and irregularly-shaped spaces (asterisks). Triangular (white asterisk), quadrilateral (purple asterisks) and pentagonal (yellow asterisk) spaces in an extracellular microfilament network are shown.
• a large bundle (cyan arrow) is formed by several long, parallel, and twisted extracellular microfilaments. The area framed with white dashes is enlarged and shown in (b).
• FIG. 5A shows a merged fluorescence image that extracellular microfilaments (ECMF, red arrows) connect via extracellular microfilament connection nodes (EMCN, purple arrows) with regularly and irregularly-shaped spaces (asterisks). Triangular (white asterisk), quadri
• FIG. 5B shows two extracellular microfilaments twist and form a twisted bundle with two rings (blue arrows).
• FIGS. 6A-B show fluorescent images of extracellular microfilaments forming superlarge, porus, multilayered and dense networks.
• FIG. 6A shows fluorescence images of a part of a superlarge, continuous, extracellular microfilament network made up of multiple networks in a 10cm dish.
• HMECs with EGFP-PMCA2 overexpression were transplanted onto the Matrigel matrix surface and cultivated for 80h. Two cell masses and the extracellular microfilament network surrounding them are shown.
• FIG. 6B shows the enlarged image of the white -framed area in (FIG. 6A).
• the extracellular microfilament networks of the two cell masses widely connect and form a continuous superlarge network complex with no interruption.
• the focused top layer and the unfocused lower layer in the same superlarge network are shown.
• There are large quantities of small pores (white asterisks, pore size range: 0.1 ⁇ 5 ⁇ ) throughout the multi-layered extracellular microfilament network.
• Scale bar 20 ⁇ .
• FIG. 7 shows an image of cells migrating on the surfaces of the superlarge extracellular microfilament meshes.
• HMECs were transplanted onto the Matrigel matrix surface and cultivated for 1 lOh.
• H&E image shows that several HMEC cell masses are disassembled (blue arrows; DCM, disassembled cell masses).
• the individual cells (indicated by red arrows), which have detached, migrated, and left the cell mass sites, migrate on the surfaces of the superlarge extracellular microfilament network (but not in the large hole where the Matrigel surface is exposed). These mobile cells are of variable morphologies but none possess the sphere morphology.
• White arrows non-disassembled cell masses.
• Scale bar ⁇ .
• FIGS. 8A-C show a schematic diagram and images of generation of 500 cm 2 , ultra- large scale and cell-less extracellular microfilament assembled network complexes.
• FIG. 8A shows a schematic diagram of generation of artificial, 500 cm 2 superlarge and HMEC mass- engendered microfilament network complex.
• HMECs are implanted on Matrigel matrix layers in the cell culture media. HMECs migrate, aggregate, proliferate, and form cell masses. Subsequently, the cell masses generate long, branched, and membrane-enclosed extracellular microfilaments. Nested extracellular microfilaments form networks exterior to and surrounding the cell masses.
• the extracellular microfilament networks connect and form a 500 cm 2 continuous, ultra-large lattice covering the entire Matrigel surface.
• a 500 cm 2 ultra-large-scale, cell-less, extracellular microfilament network (EMN, red arrow) is produced.
• FIG. 8B shows a hematoxylin and eosin (H&E) staining image of part of a superlarge continuous HMEC extracellular microfilament network (EMN, red arrows) with cell masses (CMs, blue arrows) on the Matrigel matrix surface of a 500 cm 2 plate. There is no large (diameter > 20 ⁇ ) round hole caused by the disassembly of extracellular microfilament network at this stage.
• FIGS. 9A-C show that acellular ultra-large microfilament lattices (AUMLs) promote the reepithelialization and healing of deep second degree thermal burn wounds and the generation of keratinocyte tunnels and EMNs in the reepithelialized epidermis.
• FIG. 9B shows keratinocytes forming large tunnels in the reepithelialized epidermis (REED).
• a representative immunofluoresence image shows that there are multiple keratinocyte tunnels (KTs) in the RE-ED in the AUML treated wounds.
• the large KT1 lumen contains many cells of different types.
• the KT2 lumen harbors keratinocyte EMNs.
• the framed KT1 and KT2 areas are enlarged in FIGS. 11B-C, respectively.
• Dermis (DM) and hair follicles (HF) are shown.
• Scale bar 10 ⁇ .
• FIG. 9D The enlarged area in FIG. 14B.
• a representative immunofluoresence image shows that a large keratinocyte tunnel (KT, labeled with purple dashed lines) lumen contains large keratinocyte EMNs (KEMNs), which supply scaffolds and environments for cell migration and behavior (white arrows).
• KEMNs range arrows
• irregular holes purple asterisks
• FIGS. 10A-B show AUMLs facilitate the re-epithelization and healing of second degree thermal burn wounds and allow the generation of keratinocyte tunnels.
• FIG. 10A shows representative images of second degree thermal cutaneous burn wounds with/without treatment of Mepiform or AUMLs in mice at different day post-burn (dpb).
• FIG. 10B shows representative H&E images of sectioned specimens of the burn wounds from each mouse group at 14 dpb.
• the blue-framed area is shown in FIG. 11 A.
• Reepithelialized epidermis (RE-ED) and dermis (DE) are shown.
• FIGS. 11A-C show keratinocyte tunnels and EMNs provide avenues for cell transport in the reepithelialized epidermis in the AUML treated wounds.
• FIG. 11 A shows enlarged area in the framed area in FIG. 10B.
• KT1 keratinocyte tunnel-1
• RBC red blood cells
• KEMN keratinocyte EMN fragment
• KEMN keratinocyte EMN
• FIG. 11B shows enlarged area in the framed area (in white) in FIG. 9B.
• keratinocyte tunnel-1 KTl
• red blood cells purple arrows
• ON orthochromative normoblast
• FIG. 11C shows enlarged area in the framed area (in orange) in FIG. 9B.
• KEMN keratinocyte EMN
• the membrane-enclosed and extracellular microfilament composed keratinocyte EMN (KEMN, orange arrows) is shown.
• a nucleated cell (white arrow) migrates in the keratinocyte EMN.
• Scale bar is 20 ⁇ in (a) and 10 ⁇ in (B-C).
• FIGS. 12A-C show keratinocyte tunnels and EMNs provide pathways for cell transport and migration in the reepithelialized epidermis in the AUML treated wounds.
• FIG. 12A is a representative H&E staining image shows that there are two keratinocyte tunnels (KTl and KT2) in the reepithelialized epidermis in the wounds with AUML treatment. The framed areas (KTl and KT2) are enlarged and shown in (b) and (c) respectively. Reepithelialized epidermis (RE-ED) and dermis (DE) are shown.
• FIGS. 12A-C show keratinocyte tunnels and EMNs provide pathways for cell transport and migration in the reepithelialized epidermis in the AUML treated wounds.
• FIG. 12A is a representative H&E staining image shows that there are two keratinocyte tunnels (KTl and KT2) in the reepithelial
• FIG. 12B shows many mature enucleated red blood cells (RBCs, black arrows) migrate in the large keratinocyte EMN (KEMN) in the KTl lumen.
• FIG. 12C shows two nucleated cells migrate in the large keratinocyte tunnel-2 (KT2).
• FIGS. 13A-C show keratinocyte EMNs supply scaffolds for cell migration and behavior in the keratinocyte tunnel lumen and keratinocyte EMNs disassemble.
• FIG. 13 A is a representative H&E staining image shows that keratinocyte EMN complexes are formed in the large keratinocyte tunnel lumens in the reepithelialized epidermis in the AUML treated wounds.
• Reepithelialized epidermis (RE-ED) and dermis (DE) are shown.
• the framed area is enlarged and shown in (B).
• FIG. 13B shows the enlarged area in (A).
• KEMN keratinocyte EMN
• KT large keratinocyte tunnel
• the black arrows show the keratinocyte EMN fragments and the black asterisks show the big holes (or cross sections of big channels) caused by the disassembly of keratinocyte EMN complex.
• C The farmed area is enlarged and shown in (C).
• FIG. 13C shows the archeology of keratinocyte EMNs.
• the keratinocyte extracellular microfilament composed EMNs (KEMN, black arrows) contains large quantities of irregular-shaped pores (green arrows) in various sizes.
• the disassembly of keratinocyte EMNs leads to many channels (black asterisks) in the keratinocyte EMNs.
• the edges (blue arrows) of the channels in the keratinocyte EMNs are shown.
• FIGS. 14A-C show keratinocyte EMN complexes build environments for cells, and keratinocyte ECMFs disassembly leads to keratinocyte EMN decomposition.
• FIG. 14A is a representative immunofluoresence image shows that AUMLs allow reepithelialized epidermis to form keratinocyte tunnel complexes harboring keratinocyte EMNs in different stages.
• Reepithelialized epidermis (RE-ED), dermis (DE) and hair follicles (HF) are shown.
• the framed area is shown in (b).
• keratinocyte tunnel complex containing three neighboring keratinocyte tunnels (KT1 , KT2 and KT3).
• the keratinocyte EMN (KEMN) in KT1 lumen is largely decomposed with a small channel (white asterisk) in the remained keratinocyte EMN.
• the majority of keratinocyte EMN in KT2 lumen is integrity while two channels (cyan asterisks) present in this EMN.
• the large keratinocyte EMN in KT3 lumen is decomposed and divided into several big or small EMN fragments. Multiple cells (white arrows) locate in the keratinocyte EMNs in the KT3 lumen.
• FIG. 14C is a representative fluorescence image shows that the disassembly of keratinocyte membrane- enclosed microfilaments leads to decomposition of keratinocyte EMNs (KEMN, orange arrows) forming channels in the EMNs (cyan arrows).
• FIGS. 15A-B show the generation of large area primary normal human cell membranes.
• FIG. 14A is a schematic diagram of large area primary normal human cell membrane generation. Cell membrane layer is about 40 nm in depth.
• the microfilament network can function as physical barriers for prevention and management of wound infection.
• Certain embodiments of the present disclosure are directed to a continuous network of cell-derived microfilaments as well as tissue engineering methods to produce large-area microfilament networks.
• the extracellular microfilaments formed under the conditions disclosed herein do not exist in multicellular organisms.
• Such microfilament networks have utilities in wound healing including, e.g., prevention of wound infection, including burn care, acute and surgical wound care.
• the microfilament networks promote cell migration and facilitate tissue regeneration.
• normal primary human epithelial cells cultured on cell matrix generate a large-area of microfilament network.
• the microfilament network is processed to remove the nuclei or DNA of the cells.
• the microfilament network is physically, chemically and/or mechanically processed to remove the cells.
• the microfilament network has utility in wound healing, e.g., as physical barrier and is applied to the wounds to prevent microorganism induced wound infection.
• the bioengineering methods produce large-area (up to 500 cm 2 ) microfilament network. Such network is useful for wound infection prevention.
• the network of cell-derived microfilaments is biodegradable and biologically compatible with the patient's tissue.
• the microfilament network is capable of excluding micro-organisms from the wound site.
• the microfilament network is semipermeable for constructing microenvironments that support the functions of the heterogeneous cells involved in tissue restoration and/or tissue regeneration.
• the large-area of continuous network of cell-derived microfilaments overcomes one of the major obstacles in the treatment and healing of patients with large-area second (or third) degree thermal burn wounds.
• ECMFs extracellular microfilaments
• nested ECMFs form superlarge extracellular microfilament networks (EMNs).
• EFNs extracellular microfilament networks
• ft2 square foot
• UMLs ultra-large microfilament lattices
• these UMLs construct an environment for cell migration.
• removing cell masses produces acellular UMLs (AUMLs) that can be used to facilitate wound repair.
• the AUMLs when applied to second degree thermal burn wounds of mice, the AUMLs allow keratinocytes to engender large tunnels in the reepithelialzed epidermis, thus providing pathways for cells and nutrients to the site of wound repair.
• Properties of these large AUMLs include biodegradability, biocompatibility, and semipermeability.
• the AUMLs containing native biochemical, biophysical, and biomechanical components that are suitable for broad use in wound repair and tissue regeneration.
• Microfilaments the main cytoskeletal polymers in eukaryotic cells, are polymerized by actin subunits and actin-binding proteins. Microfilaments are essential for cell division and cytokinesis, cell shape maintenance, vesicle transportation, signal transduction, sensing, and cell motility (Gunning, P. W., Ghoshdastider, U., Whitaker, S., Popp, D. & Robinson, R. C. The evolution of compositionally and functionally distinct actin filaments. Journal of cell science 128, 2009-2019 (2015)). Cytoskeletal actin (including ⁇ - and ⁇ -actin) assembly and depolymerization lead to microfilament network remodeling (Herman, I. M. Actin isoforms.
• ⁇ -scale of adult animal cell sizes limits the potential area of the cytoskeletal microfilament network of single cells to the ⁇ m 2 -scale (Lloyd, A. C. The regulation of cell size. Cell 154, 1194-1205 (2013), Ginzberg, M. B., Kafri, R. & Kirschner, M. Cell biology. On being the right (cell) size. Science 348, 1245075 (2015)).
• human epithelial cell masses generate superlarge extracellular microfilament networks that facilitate cell migration.
• the present disclosure provides a general strategy to engender artificial and ultra-large extracellular microfilament networks in the level of square foot (ft 2 ).
• human cell masses generate ultra-large (ft 2 ) scale mesh structures assembled by membrane-enclosed extracellular microfilaments.
• these mesh structures are used by cells as functional ECM facilitating cell migration.
• these native meshes are effective at promoting burn wound healing in mice.
• these meshes are simple, reliable, superlarge and 3D extracellular microfilament meshes (ft 2 or larger).
• these meshes are porous, native, dense, or acellular.
• these meshes could be used to facilitate wound repair and tissue regeneration.
• human epithelial cell masses generate long (up to ⁇ in length) actin polymerized microfilaments extracellularly.
• the microfilaments are membrane-enclosed.
• the cell and the cell-derived extracellular microfilaments form superlarge continuous networks, which pave paths for cell migration.
• decellularization engenders artificial, cell-less and superlarge (500 square centimeter, cm 2 ) lattices, increasing the microfilament network area up to about a billion-fold (lxl0 9 -fold) in comparison to the cytoskeletal microfilament network area in a single human epithelial cell of the equivalent size.
• the superlarge and porous extracellular microfilament meshes facilitate the reepithelialzation and healing of the second degree thermal burn wounds in mice.
• the presently disclosed methods produce ultra-large scale extracellular microfilament networks that promote cell migration and are useful for tissue regeneration and wound healing.
• subject means a vertebrate, preferably a mammal, more preferably a human.
• Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
• therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
• the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
• treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
• therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
• the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
• the term "effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results.
• the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
• the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
• the method of producing a network of microfilaments is via culturing cells in a matrix support and cell culture medium.
• the cell masses form on top of the matrix support.
• the matrix is the cell culture medium, Matrigel.
• the microfilaments of the network include actin, such as ⁇ -actin, ⁇ -actin, and actin-interaction proteins.
• the microfilaments are about 1- 1000 ⁇ , 10-900 ⁇ , 20-800 ⁇ , 30-700 ⁇ , 40-600 ⁇ , 50-500 ⁇ , 60-400 ⁇ , 70-300 ⁇ , 80-200 ⁇ , and 90-100 ⁇ in length.
• the microfilaments are branched.
• the microfilaments have about 2-10, 3-9, 4-8, 5-7 branches.
• the network further includes adhesive materials. In one embodiment, the adhesive materials associate with the microfilaments and enlarge the diameter of the microfilaments.
• the network has an area in the range of about 1 ⁇ 2 to about 500 cm 2 . In another embodiment, the network has an area of about 10 ⁇ 2 to about 400 cm 2 , about 100 ⁇ 2 ⁇ about 300 cm 2 , about 200 ⁇ 2 ⁇ about 200 cm 2 , about 1000 ⁇ 2 to about 100 cm 2 and about 1 cm 2 to about 10 cm 2 . In another embodiment, the network has a thickness in the range of about 1 nm to about 1 cm, about 10 nm to about 0.1 cm, about 100 nm to about 0.01 cm and about 1000 nm to about 0.001 cm. In one embodiment, the network is applied to an area in need of treatment as a single layer. In another embodiment, multiple layers of the network can be applied to the area in need of treatment.
• the pore size of the network ranges from about 0.1 - 5 ⁇ , about 0.2 - 4 ⁇ , about 0.3 - 3 ⁇ , about 0.4 - 2 ⁇ and about 0.1 - 1 ⁇ in diameter.
• the network can include bioactive and/or bioinactive agents.
• the bioactive and/or bioinactive agents include integrins, adhesion receptors, and membrane proteins.
• the bioactive agents are therapeutic drugs including antibodies and microorganism inhibitors.
• the network is present on a matrix support.
• the matrix support is biodegradable.
• the network is present on a Matrigel matrix support.
• the microfilament source regions include eukaryotic cells with or without genetic modification.
• the present invention provides a method for treating a medical condition via applying the microfilament network of to an area in need of treatment.
• the medical conditions relate to many types of wounds known to a skilled in the art, including but are limited to wounds, acute wounds and chronic wounds, burn wounds, thermal burn wound, chemical burn wounds, and electric burn wounds.
• the microfilament network is applied with the matrix.
• the method further includes separating the matrix substrate from the continuous extracellular microfilament network and the microfilament network is applied without the matrix.
• agents and compositions described herein according to the various methods of the invention may be achieved according to a variety of methods.
• the agents and compositions of the invention can be administered by any suitable means, e.g., parenteral, intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracistemal, intraperitoneal, buccal, rectal, vaginal, intranasal or aerosol administration.
• Administration may be local, i.e., directed to a specific site, or systemic.
• Administration may also be effected by, but not limited to, direct surgical implantation, endoscopy, catheterization, or lavage.
• compositions of the invention may be flowed onto the tissue, sprayed onto the tissue, painted onto the tissue, or any other means within the skill in the art.
• compositions of the invention applied during surgery may be incorporated into a suitable matrix.
• compositions of the invention applied during surgery may be implanted in a patient at the site of a wound where re-epithelialization is desired.
• compositions of the invention may be administered in or with an appropriate carrier or bulking agent including, but not limited to, a biocompatible oil such as sesame oil, hyaluronic acid, cyclodextrins, lactose, raffinose, mannitol, carboxy methyl cellulose, thermo or chemo-responsive gels, sucrose acetate isobutyrate.
• a biocompatible oil such as sesame oil, hyaluronic acid, cyclodextrins, lactose, raffinose, mannitol, carboxy methyl cellulose, thermo or chemo-responsive gels, sucrose acetate isobutyrate.
• concentration of the drugs/compounds described in the compositions of the invention will vary depending upon a number of factors, including without limitation the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration.
• the preferred dosage of drug to be administered also is likely to depend on variables including, but not limited to, the type and extent of a disease, tissue loss or defect, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the presence and types of excipients in the formulation, and the route of administration.
• the therapeutic molecules of the present invention may be provided to an individual where typical doses range from about 10 ng/kg to about 1 g/kg of body weight per day; with a preferred dose range being from about 0.1 mg/kg to 100 mg/kg of body weight, and with a more particularly preferred dosage range of 10-1000 ⁇ g/dose.
• HMECs Normal primary human mammary epithelial cells
• HMECs normal primary human mammary epithelial cells
• MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM CloneticsTM MEGMTM Mammary Epithelial Cell Growth Medium plus SingleQuotsTM Kit package
• HMECs were cultured in MEGM BulletKitTM with/without the Matrigel matrix layers at 37 °C in humidified 5% CO2 atmosphere.
• EGFP-hPMCA2z/b # 47584
• mCherry- -actin # 54967 plasmids were ordered from Addgene.
• Anti-y-Actin gamma Actin, monoclonal, abl23034
• Anti-pan-Cadherin polyclonal, abl403378 antibodies were ordered from Abeam.
• MatrigelTM Membrane Matrix CB -402314
• Corning® Matrigel® Basement Membrane Matrix, Phenol Red- Free, *LDEV-Free (Product #356237) were purchased from Corning.
• Corning® 500 cm 2 Square TC-Treated Culture Dishes (Product #431110) were ordered from Corning.
• Mepiform® (a silicone membrane for wound care, Warner, P. M., Coffee, T. L. & Yowler, C. J. Outpatient burn management.
• the Surgical clinics of North America 94, 879-892 (2014).) was ordered from MOlnlycke Healthcare.
• the 6-week-old female mice (Strain name: BALB/cf) were ordered from the Jackson Laboratory. Animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Harvard Medical School.
• the MatrigelTM Membrane Matrix was thawed at 4 °C overnight.
• the Matrigel layers (20-30 ⁇ in depth) were prepared in pre-chilled 6-well-plates, 10 cm dishes or 500 cm 2 square dishes, followed by gel for 20 minutes at 25 °C in humidified 5% CO2 atmosphere.
• the Matrigel layers were prepared with Phenol Red-Free Matrigel on the VWR-Micro covers in 6-well-plates.
• HMECs were plated on the Matrigel layers and cultured in the MEGM BulletKitTM media at 37 °C in a humidified atmosphere of 5% CO2.
• the extracellular microfilaments were developed from 48-1 lOh after cell culture. Plasmids of EGFP- hPMCA2z/b (Addgene, #47584) and mCherry- -actin (#54967) were transfected into HMECs using lipofectatine® 2000 (Life Technologies, #11668027) according to the manual. Anti- ⁇ - Actin (gamma Actin, monoclonal, abl23034) and Anti-pan-Cadherin (polyclonal, abl40338) antibodies were ordered from Abeam. Two days after transfection, the transfected cells were plated on the Matrigel matrix layers (1 x 10 3 cells per well of 6-well-plate) in the indicated media.
• H&E Hematoxylin and eosin staining of cells was performed after cell fixation.
• Samples with VWR-Micro covers were transferred onto glass slides followed by imaging acquisition. Imaging acquisition. Phase contrast images were taken with a Nikon TMS inverted phase contrast microscope and a Nikon Coolpix auto 4300 digital camera. Fluorescence images of fixed cells were taken with an 80i upright microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20x or 40x lens and MetaMorph image acquisition software. Hematoxylin and eosin staining images of fixed cell or tissue sections were taken with 80i upright microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20x or 40x lens and NIS-Elements acquisition software.
• HMECs The fixed HMECs were then postfixed for 30 min in 1% osmium tetroxide (Os04)/1.5% potassiumferrocyanide (KFeCNe), washed in water 3 times, and incubated in 1% aqueous uranyl acetate for 30 min. This was followed by 2 washes in water and dehydration a gradient of alcohol (5 min each; 50%, 70%, 95%, 2 x 100%) (Basler, M., Pilhofer, M., Henderson, G. P., Jensen, G. J. & Mekalanos, J. J. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182-186 (2012)).
• Os04 osmium tetroxide
• KFeCNe potassiumferrocyanide
• HMECs and ultra-large scale extracellular microfilament networks in the 500 cm 2 dishes were fixed with 4% paraformaldehyde for 10 min, followed by 3 washes with lxPBS and H&E staining.
• the cell masses and cells were removed with pipette tips (1 mL or 100 ⁇ ) and a Nikon TMS inverted phase contrast microscope followed by three washes with lx PBS.
• cells and superlarge extracellular microfilament networks were fixed with 0.5% KMn04 (in lx PBS, pH 7.1, for lipid stabilization, Zhao, S.
• Double immunohistostaining analyses The cutaneous wounds were excised, fixed with HistoChoice® MB fixative (Amresco), and embedded in paraffin. The sectioned specimens (5 ⁇ in depth) were subjected to double immunohistochemistry staining with anti- pan-Cadherin antibodies (Abeam, abl40338, 1:200; Life Technologies, # 982425, Alex Fluor®488 goat anti-rabbit IgG (H+L) secondary antibody, 1 : 1000), anti-y-actin antibodies (Abeam, abl23034, 1 :200; Life Technologies, Alex Fluor®568 goat anti-rabbit IgG (H+L) secondary antibody, 1 :1000), and 4',6-diamidino-2-phenylindole (DAPI, 1 : 1000). Fluorescence images were taken with a Nikon 80i upright microscope with a 20x/40x/60x lens. All images were obtained using MetaMorph image acquisition software.
• HMECs Normal primary human mammary epithelial cells
• 3D Matrigel matrix-culture media mixture gel layer
• the individual HMECs did not exhibit irregular shapes, but consistently presented a spherical morphology, with a minimal surface area remaining in contact with the Matrigel (FIGS. 1A- B). This indicates that the Matrigel is an unfavorable environment for cell adhesion, attachment and spreading.
• the HMECs migrated, aggregated, proliferated, and formed compact cell masses with multiple stacked cells maintaining no contact with the Matrigel matrix (FIGS. 1C-D).
• Plasma membrane calcium- transporting ATPase-2 functions as a calcium extrusion pump that removes Ca 2+ from cells (See, Street, V. A., McKee- Johnson, J. W., Fonseca, R. C, Tempel, B. L. & Noben- Trauth, K. Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice. Nature genetics 19, 390-394 (1998)) and regulates the Ca 2+ content of a number of cell types, including mammary epithelial cells (See, VanHouten, J. et al.
• PMCA2 regulates apoptosis during mammary gland involution and predicts outcome in breast cancer. Proceedings of the National Academy of Sciences of the United States of America 107, 11405- 11410 (2010)). Thus, HMECs were transiently co-transfected with plasmids encoding enhanced green fluorescence protein (EGFP)-tagged PMCA2 (EGFP-PMCA2). Upon fluorescence imaging, EGFP-PMCA2 was found to be distributed throughout the plasma membranes of cells and along the surfaces of a large portion of the nested microfibers surrounding the cell masses. This suggests that the cell masses vigorously produce large quantities of membrane-enclosed extracellular microfibers (FIG. 2A) that connect the cell masses. The long and short extracellular microfibers densely connect and thus constitute a continuous network (FIG. 2A).
• EGFP enhanced green fluorescence protein
• the porous (multilateral pores), extracellular microfilament assembled networks then connect, forming a superlarge, continuous, porous extracellular microfilament network surrounding the cell masses (FIGS. 2A-B).
• the extracellular microfilaments possess potent ability for branching morphogenesis (FIGS. 2A-B and FIG. 3).
• the number of extracellular microfilaments increased rapidly, forming dense networks quickly (FIG. 4A-E).
• FIG. 4A-E The above data suggests that these superlarge meshes of persistent, long, highly branched, membrane- enclosed extracellular microfilaments, which do not exist in multicellular organisms to our current knowledge, are created here for the first time.
• HMECs were continuously cultured for 110 h after the implantation of cells on the Matrigel layers.
• the cell masses began to disassemble, and the individual cells detached, migrated, left the cell mass sites, and either remained on or traveled along the surfaces of the superlarge EMN, avoiding the Matrigel surfaces in the large round holes which are caused by the disassembly of EMNs (FIG. 7).
• the individual cells originating from the disassembled cell masses possessed irregular morphologies, with no spherical cells being observed (FIG. 7).
• HMECs To examine whether the cell masses can effectively develop ft 2 -scale EMNs, we implanted HMECs on the Matrigel layer of 500 cm 2 culture plates. The cell masses generated ultra-large EMNs that covered the entire 500 cm 2 Matrigel surface (FIGS. 8A-B), demonstrating the cell masses' extraordinary capacity to produce ultra-large scale EMNs, which suggested potential applications for wound healing if the cells are removed. The removal of xenogeneic and allogeneic cellular genetic materials via decellularization results in the production of minimally immunogenic native structures for use in tissue regeneration 26 .
• Re-epithelialization and wound healing is a challenge for patients with large-area second (or third) degree thermal burn wounds.
• One of the major obstacles to treatment and healing is the absence of large-area continuous materials with the required characteristics: the material must be degradable, biologically compatible with the patient's tissue, capable of excluding micro-organisms from the wound site, and semipermeable in order to construct microenvironments (See, Heng, M. C. Wound healing in adult skin: aiming for perfect regeneration. International journal of dermatology 50, 1058- 1066 (2011)) that support the functions of the heterogeneous cells (See, Taylor, G., Lehrer, M. S., Jensen, P. J., Sun, T. T.
• keratinocytes generates EMNs in the keratinocyte tunnel lumens and diverse cells (including red blood cells) migrate in the keratinocyte EMNs, demonstrating that keratinocytes engender EMNs in vivo providing scaffolds for cell migration and other behavior (FIG. 9D and FIGS. 11A-C, 12A-C and 13A- C and 14A-C).
• Keratinocyte EMNs disassembled and cells filled the keratinocyte tunnels, which coincides the facts that keratinocyte tunnels and EMNs disappeared in the reepithelialized epidermis at the later stage, suggesting that AUMLs create appropriate environments for the orchestrated generation and decomposition of keratinocyte tunnels and EMNs (FIG. 9D, FIG. 10B and FIGS. 11A-C, 12A-C and 13A-C and 14A-C).
• the ft 2 -scale AUMLs which are completely generated by human cells, naturally employed by cells as scaffolds and environments for cell migration and other behavior, decellularized while keeping the native biophysical, biochemical, and biomechanical signals intact, enabled the application of AUMLs for the facilitation of wound repair.
• the characteristics of cell membrane-enclosed extracellular microfilaments allow the AUMLs to be used as biodegradable, biocompatible, semipermeable, and minimal immunogenic biomaterials for the facilitation of wound repair and tissue regeneration.
• the potential for creating very large area-AUMLs allows them to be easily tailored to wounds of any size.
• AUMLs promote the generation of large keratinocyte tunnels providing pathways for cells and nutrients, and the production of keratinocyte EMNs supplying scaffolds for cell migration and behavior in vivo.
• the keratinocyte tunnels and EMNs fulfill the high demand for cells, soluble growth factors and nutrients in areas of the epidermis where cell migration, proliferation, differentiation and stratification are taking place as during tissue repair.
• biodegradable, biocompatible, semipermeable and infinite AUMLs with their native biophysical, biochemical, and biomechanical signals will be applied as a method of treating a broad spectrum of wounds and facilitating tissue regeneration, as large-area native meshes facilitate cue-guided cell migration, proliferation and differentiation in developing highly organized tissues.
• Data provided herein show that a previously unrecognized extracellular microfilament network, produced from cells grown in Matrigel, facilitates cell migration.
• the present disclosure contemplates cell-less and ultra-large scale (ft 2 scale) extracellular microfilament networks that facilitate the re-epithelialization and healing of second-degree thermal burn wounds.
• Normal primary human epithelial cells cultured on cell matrix generate a large-area (up to 500 cm 2 ) of primary normal human cell membrane without nuclei or DNA, which can be applied to the wounds and acts as physical barriers to prevent micro-organism induced wound infection.
• the engineered large area cell membrane- matrix complex layer can effectively prevent infection by micro-organisms, including bacteria, fungi and viruses.
• MatrigelTM matrix layers (Yi, T., Kabha, E., Papadopoulos, E., and Wagner, G. (2014) 4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance, proliferation and metastasis.
• Primary normal human mammary epithelial cells HMECs
• HMECs Primary normal human mammary epithelial cells
• the large area native primary normal human cell membranes generated by this method have the characteristics including: 1) native cell membranes without nucleus with super-large area (up to 500 cm 2 or more); increase billion-fold (10 10 -fold) in area compare to a single cell area; 2) double native primary normal human cell membrane layers; 3) no genetic materials of DNAs; 4) native lipid membrane with native membrane proteins; 5) easy degradation of cell membrane and Matrigel matrix; amd 6) wound infection prevention.

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